Biochem Exam #2

study of enzyme kinetics begin in —- when —- studied —-

1902 when Adrian Brown was studying sucrose hydrolysis

Adrian Brown enzyme kinetics info

rxn: sucrose + water (enzyme of B-fructofuranosidase/invertsase) yeileds glucose + fructose

when [sucrose] > > than [enzyme] the reaction rate is independent of [sucrose], ie the rxn is zero-order with respect to sucrose

so he proposed a two step rxn mechanism in which the substrate forms a complex with the enzyme that then decomposed to produces P and free energy

the complex with the enzyme (ES) is not the same as the transition state

when substrate concentration is much greater than enzyme conc then the enzyme is saturated so that all the enzyme is in ES form

under this condition then the second step of the rxn is rate limiting as more substrate does nothing to the overall reaction rate

michaelis and menton general

used the knowledge from brown’s experiments to mathematically describe rxn rate of the scheme E + S yields ES yields P + E

when S conc is sig. greater than E conc then the rxn rate of product formation from ES is a first order process

-however, this expression does not completley describe catalyzed rxns as all enzymes are not always saturated. Rather, the change in the concentration of the ES complex is the rate that it is formed minus the rate that it breaks down.

Assumption 1:

Pre-Equilibrium

assume that k-1 >> k2 so that the first step reaches equilibrium

Ks is a dissociation constant for the first step

This is why ES is known as the Michaelis complex

PROBLEM: this assumption is not often correct (but it is reasonable for most cases according to prof)

Assumption #2

Steady State

under physiological conditions, common for [S] > > [E], the [ES] remains nearly constant until substrate is consumed… in other words ES maintains a steady state

note that a rxn at steady state is not a equilibrium as conc. S and conc P are rapidly changing when conc ES and conc E are constant

total concentration of an Enzyme equation

[E]t = [E] + [ES]

unfort for all the equations and stuff… i have no idea so go read the slides ok

ok

what is initial velocity

experiemtnally: velocity measured before more than ~10% of substrate has been converted to product

The [S] that gives 50% Vmax yields the Km

okay so basically small km means the enzyme achieves maximal catalytic efficiency at low [S]

Km depends on

identity of enzyme, substrate, pH, and temperature

when [S] < < Km then

rxn rate varies linearly [S]

when [S] > > Km then

enzyme is saturated so that Vo=Vmax

when k2 < k-1 then….

Km represents Kd

lineweaver-burk plot

plots of 1/V0 vs 1/[S] will be linear, with slope = Km/Vmax and y intercept equal to 1/vmax and x intercept = -1/Km

usually you collect data from 0.5-5 Km

kcat

is always a first-order rate constant, so units of s-1

it is also referred to as the turnover number, which represents the number of reactions per second under fully saturating conditions

under physiological conditions the [S]/Km ratio is usually 0.01-1 thus

kcat/Km is the apparent second-order rate constant

Kcat/Km is a measurement of

catalytic efficiency (or specificity constant) fan an enzyme for a particular substrate

the higher the ratio, the more efficient that enzyme is with that substrate

it is a metric to compare different substrates for teh same enzyme or different enzymes with the same substrate

note that km is = to the substrate dissociation constant (Kd) only if k-1 > > k2

okay

since overall catalysis can be no faster than the rate constant k1 for substrate binding…

catalysis happesn as fast as the enzyme and substrate collide into eachother

the upper limit for any 2nd order rate constant in biochem is the

diffusion-controlled limit 10^8 - 10^9 M-1s-1 … simply how fast two things can collide into each other in water

under steady state conditions…

mechanistic details of a reaction cannot be determined

aka steady state conditions cannot determine microscopic rate constants or the number of intermediates by examining only the concentrations of substrate and/or products

competitive inhibition - reversible

prevents/weakens substrate binding without affecting catalytic activitiy

molecule binds to teh active site… can be overcome by adding more substrate

uncompetitive inhibition - reversible

reduces catalytic rate without weakening substrate binding affinity

molecule only binds to an ES complex. only binds to the union of E and S

mixed inhibition - reversible

elements of both, affects substrate binding and catalytic activity

molecule binds to the enzyme at allosteric site either in the presence or the absence of the substrate and reduces enzyme efficiency

irreversible competitive inhibitors

form a covalent bond with an active-site residue to irreversibly inactivate enzyme

methods of enzyme regulation: control of localization

sequester enzyme away from substrate until the time is right

methods of enzyme regulation: allosteric modulation

just like allosteric modulators for binding affinity

enzyme has at least 2 binding sites, conformational change from allosteric modulator changes enzyme activity

often used as mechanism of positive or negative feedback

methods of enzyme regulation: covalent modification

may enhance or reduce enzyme activity

can be reversible or irreversible

may have multiple sites

this is a way to integrate multiple signals

creates a gradient of signals, not just binary on/off

focus on phosphorylation: the most abundant, common, and important type of covalent modification to regulate enzymes

-kinases: add phosphate group

-phosphatase: remove phospahte group

technically cutting up a protein is also considered a covalent modifiaction, called proteolysis

zymogen

an inactive molecule that is converted into an zyme when activated by another enzyme

Alexander Todd

hydrolyzed precipitates and found nucleotides

-proposed major components of nucleic acids were base, sugar, phosphates

Franklin

-isolated a small piece of nucleic acid

-crystallized it and discovered that there was symmetry in DNA

-she gave a lecture and Francis + Crick were in the audience

Francis + Crick

-build cardboard model of DNA… double helix

DNA and RNA are polymers comprised of

nucleotides

-there is a ton of non coding dna that we do not know what it does but its probably involved witth regulation

nucleotide vs nucleoside

The fundamental difference between a nucleoside and a nucleotide is the presence of a phosphate group.

• Nucleoside = Nitrogenous Base + Pentose Sugar

• Nucleotide = Nitrogenous Base + Pentose Sugar + Phosphate Group (a nucleoside with one or more phosphate groups attached)

3 groups of nucelotide

nitrogenous base, pentose sugar, phospate

Nitrogenous Bases

either purine or pyrimidine

purine = 2 fused rings

pyrimidine = 1 ring

bases are planar, aromatic, heterocyclic molecules

each atom is identtified by a number

purine and pyrimide iupac number system

difference between dna and rna bases

RNA has uracil instead of thymine

random ahh note he about pyrimidines

N9 position of purines and N1 position of pyrmidines are linked to sugar moiety

where do theh bases link

at the single rings they all link via Nitrogen 1

in double ringhs they all link via the N in the 5 membered ring

Japense space craft story

all five nucleobases were discovered from an asteroid

nearly equal amounts of purines and pyrimides (shows that it is not random)

this widespread presence throughout the solar system reinforces the hypothhesis that carbonaceous asteroids contributed to the prebiotic chemical inventory of early earth

nitrogenus bases - structure and pH

nitrogenous bases have different tautomers, structural isomers that differ in location of hydrogens

tautomerization (proton shiftt) depends on pH

Amino and keto forms are favored att physiological pH

potential source of chemical diversity dependiing on micro-enviroment

adenine tautomerization pka

3.6

cytosine tautomerization pka

4.2

guanine tautomerization pka

9.4

thymine tautomerization pka

9.5

ribose vs deoxyribose

ribose = OH, RNA

deoxyribose = H, H

sugar is chemically and structturally different in RNA and DNA

carbon atoms identified by a number, followed by the prime symbol (‘)

functions of nucleotides

-carry chemical energy in cells

-chemical potential energy of ATP is used when phosphate groups are transferred to another molecule (thermodynamic coupling)

-nucleotides can be second messengers

-cylic AMP is produced in response to hormones and other chemical signals

DNA and RNA are polymers of nucleotides

-glycosidic bond between base and sugar

-phosphate diester = point of stability in the DNA

-must nuecleotides in cells exist in polymeric form (ie as chains of DNA and RNA)

-Phosphodiester bonds link successive nuecloties in DNA and RNA

-chain has polarity ie 5’ vs 3’

-both DNA and RNA are negative due do the phosphates

structure of DNA

discovered in 1953

exists as a right handed double helix in antiparallel strands

complementary

bases occupy core of helix while sugar-phospate groups are on the periphery

base pairs form hydrogen bonds to each ttoher

A-T/U and G-C

major and minor groove, grooves expose edge of base pairs, providing points of recongition by proteins

need to look at the slide that has H bond donor/acceptors

usually the N and O are H bond accepttors and the H/NH are H bond donators

a straight double bond O can accept two H bonds but it often doesnt because it is limited by helical structure

A/B/Z form

DNA and RNA form double helixes duh, but there can be A form or a B form

A = C3-endo

B= C2-endo

the Z form is very rare because pyridines and primidines must be alternating to form it

RNA Motifs

stem loop, bulge, single base bulge, internall loops, mismatch, thhree way and four way

GU wobble base pair is common in RNA structure

computer software can now predict any secondayr structure, but 3D structure sttill needs experimentation to be solved

how do you determine 3D structure of RNA

either xray or NMR

RNA function

-RNA has capacity for information storage and catalysis

catlayic RNA or ribozymes

The 2’-OH is crtical for RNA catalysis

in line attacks occur under basic conditions

RNA is iinherently unstable…evolution chose to have RNA for certain purposes…by choosiing to take hydroxy out of DNA then it is less leakly to cleave the backbone which iis less chance for modification but increased stability

RNA world hypothesis

explains how an RNA-based life existed first

RNA forms- ribozyme catalyzes RNA replicattion - RNA catalyzes proetin synthesiis

mutations in DNA

result in DIVERSITTY but in the shortt tterm… result in disease

both DNA and RNA have multiple levels of structure

including the supercoil

Transcription

DNA to mRNA

mediated by RNA polymerase and associated proteins

position of inition and terminattion determined by DNA sequence

speciific proteins bind to initian and termination sites to interact wiith RNA polymerase to start or stop transcription

3 major subtypes of RNA

messenger RNA = message which is translated into protein

ribosomal RNA = structural component of ribosome (massive)

Ribosome = is composed of protein and rRNA), two subunits

reserch techniquesa involving nucelic acids

PCR tto amplify DNA

southern blott = ligattes with DNA

northern blot = detects RNA

Western blot = antibody based approach

Crispr = gene editing

Lipids intro

one of the four biomoleculues class

however unlike the other biomolecules they are nott polymeric, instead they aggregate

used in cells to form bilayers, energy stored in hydrocarbon chains, intra and intercellular signaling events

oligosaccharide

in diseases oftten times these sugars on the outside of the cell have been altered and could be potenttial ttarget for vaccines

peripheral protein

no anchor, uses electrostatic forces to hold near the surgace

peripheral protein

covantley linked to lipid membrance via lipid anchor

lipid membranes serve to

compartmentalize components of cells, delinate in/out

difine subcellular organelles in eukaryothes

segrate different metabolic actibity

selective permeability

separations of regions with different chemical requirements

formarion of chemical gradients

transport of singalinig molecules

know the story of mitochondria

ok

lipids intro continued

lipids defiined as substances of biological orgin that are solubel in organic solvents but are only spariingly soluble in water

lipiids also serve as long-term energy storage reservoirs (contrast with starch/glycogen for short term)

major classes of lipids

fatty acids = carboxylic acids with long chani hydrocarbons

triacylgylcerols- fatty acid triesters of glycerol

glycerophospholipids = major lipid componets of biological membranes

sphingolopids = deriviteves of amino alchhohol spingosine and dihydrospingoshine, ceramids are the parent compound

sterols = mostly eukaryotic in orgiin

acyl

know

acetyl

know

glycerol

know

ethanolamine

know

choline

know

fatty acid info

are defined as carboxylic acids with long chain hydrocarbon side groups

biologically common fatty acids have 14-20 carbons but C16 and C18 predominiate

they are usually even nuberewd because theyre consttructed 2 carbons at a time

less than 50% are unsaturated (contain at least one double bond) but most of these are polyunsaturated (cotain more than one double bond)

first double bond of an unsaturated fatyt acid occurs between its C9 and C10 carbonds and tend to occur every third carbon

full saturation would mean no double bonds

double bonds in naturally occuring fattty acids are almost always cis

saturated fatty acids

highly flexible bc of free rotation around single C-C bonds but the extended confromation is most populated because iit minimazes steric hindrance between neighboring methhyleene gorups

each cis double bonde in an unsaturated fatty acid places a 30 degree kink in the structure

lipids associate in water due to the hydrophobic effect

lipid packing thus depends on saturation of the lipeids

unsaturated fatty acids pack togehtter less efficienty than saturated fatty acids because redued van der Waals interactions of unsaturated fatty acids cause melting pointts ot decrease

fluiding increases with degree of unsaturation, rigidity increases with degree of saturation (higher mp)

saturated fatty acids can pack more tightly, therefore, theri melting poinst are highher

unsaturated fattty acids are more fluid, less packed, and have lower MP

triaclyglcerols

fats and oils in plants/animals consist of mixtures of ttriacylglercols which are fatty acid triesters of glycerol which serve as energy reservoirs

differ based on identify and location of 3 fatty acid cains

simple triacyclglcyerols contain only one type of fattty acid, while mixed can contain two or tthree different types of fatty acids and are more common

wax

wax is along chain fatty acid combined with a long chain alchohol

the fatty acid and the alchohol can both be either saturated or unsaturatted

glycerophospholipids

are the major lipid components of biological membranes

consist of flycerol-3-phosphatte esterified at C1 and C2 with fattty acids and phospahte group linked tot a polar head group

they are amphiphillic molecules… nonpolar tails and polar headgroups

fatty acid side chains vary between unsaturated and saturated

head group is commonly derived from polar alchohols

they are named according to head group

glycerophospholipids naming

according ot head group

net charge varies depending on head group identify - contrubutets ot hte properties of membrane structure

glycerophospholipids

memporize all those molecules okayyyyy bye

sphinogolipids

another major lipid membrane component, particular prevalent in neural tissue

cermaide derivitives of sphinogosine -take sphinogosine and attach a fatty acid to its primary amine, then a polar group to its C1 hydroxyl, and you get a ceramide

sphingomyelines

cermaides with phosphocholine or phoshoethanolamine as a head group

most common of the sphinogolipids

found predominatly in the myeline sheath around nerve fibers

cerebroside

ceramides with a sugar head group

unlike phospholipids, cerebrosides lack phosphate groups and are nonionic

galatocerbrosides are most prevalent in brain cell membrances

gangliosides

ceramides with a complex sugar head group tthat contains at least one sialic acid residue

primarily components of cell-surface membrancs, about 6% of brain lipids

most complex group of sphingolipids, complex carbohydratte head groups extend beyond tthe cell membrane

some inheritted human diseases cause abnormal accumulation of membrane lipids

Niemann-Pick is a caused by a rare defect in the enzyme sphingomyelinnase which cleaves phosphocholinen from spingomyeline

spinogomyeline accumlates in the brain, spleen, and liver, eventually causing mental handicap and early death

steroids

deriviatives of four fused nonplanar rings

major component of animal cell membranes, acting mostly as membrane rigidifying molecules

most abundant steroid in animals

has a polar head, a rigid fused ring structure, and a hydrocarbon tail

has complex effects on membrane fluidity

is the metabolic precurser of steroid hormones

-fused rings are much less flexible than lipids

-weakly amphiliplic

acquire through diet but you can also produce

steroids history

mass spectrometry documented the presence of cholesteroids (precursers) in the first animal the dickinsonia

-the surrounding sediments had stigmasteroids

steroids precurer

cholesterol

steroids are more polar than cholesterol because no alklyl chain attached to ring D, important role in gene expression

certain vitamins are lipids

ex vitamine E, vitamin K, etc….

natural pigments are also lipids

ex = red and yellow cardinal

other lipid functions

carotene = tranforms to retinal for light sensing

prostaglandin = signaling molecule in the inflammatory pathway

vitamin D = produced from cholesterol in the presence of UV light

lipid bilayers

micells = aggregate spherical structures

there is no water in. a micelle and micelle formation is common for single tailed amphilies

disruption of lipid bilayer results in the formation of liposomes which are solvent-filled vesicles that resemble a biological membrane

impermeable to polar molecules,

can undergo transerve or lateral diffusion

individual lipids rarely flip flop across membrane

lateral diffusion is rapid

lipid bilayer is in constant motion due to rations around C-C bonds of lipid tails

hydrophobic core begins about 15A into the membrane

lipid bilayer and phase changes

cholesterol acts as both a rigidier and plasticizer

rigidity = planar part rigidifies outer half of membrane bilayer, sterically hinders lipid diffusion at normal temp

plasticity = flexivle part of cholesterol disrupts van der waals interactions and lcose packing of alkyl chians

if too little cholesterol then bilayer lack protection against low temps

if too much cholesterol then lose fluidity

cholesterol concetration is higly regulated

functions of biological membranes

to define cell boundaries, define intracellular compartments, selectively permeable, to maintain electric and chemical protentials and actively transport molecules and ions

biological membranes are stiffer

in the outside of the membrane but more ligquid like in the inner portion

membrane proteins

30% of proteins are membrane proteins

interactions include: hydrophoic effect across and at the bilayer, covalent lipid anchors, interaction via transmembrane protieins, and ionic interactions via lipid surface

bacteriorrhodopsin

membrane spannign proton pump

polytopic protein

seven transmembrane alpah helices are connected by highly polar, solvent-exposed loops

its side chains are non polar

the hydropathy plots are used to predict alpha helices

transmembrane proteins use

antiparallel B sheets to form a barrel or channel across membrane

OmpF porin

a transmembrane channel is an example of a transmembrane protein that does not form alpha helices

instead, hydrogen bonding requirements met by forming an antiparallel B barrel that creates a solvent accessible central channel